Genomic organization and regulation of the human orexin (hypocretin) receptor 2 gene: identification of alternative promoters
Résumé
Orexins (hypocretins), acting via their receptors, are involved in the control of feeding behaviour, sleep, arousal and energy homeostasis. However, regulation of human orexin receptor 2 (hOX2R) gene remains unknown. We have identified four transcripts arising from alternative splicing from three exons. These exon 1 variants were designated as exons 1A, 1B and 1C on the basis of their 5' to 3'order. RT-PCR demonstrates the differential expression in various human tissues. The alternate 5'-UTRs possessed by these isoforms have different translational efficiencies, which regulate the level of protein expression. We demonstrate that the hOX2R gene is regulated by two promoters, the novel transcripts are regulated by the distal promoter located upstream to exon 1A. We demonstrated that the Ap-1 motif is critical for sustaining the basal activity of distal promoter. Analysis of the proximal promoter revealed the region regulating promoter activity contained putative binding elements including those for CREB, GATA-2 and Oct-1. Using the chromatin immunoprecipitation assay, we demonstrated that CRE, GATA-2 and Oct-1 transcription factors bind to these critical regulatory promoter elements. Mutational studies suggested that these motifs functioned independently but have a compound effect regulating hOX2R gene transcription. Furthermore proximal promoter activity is enhanced by both PKA and PKC pathway activation, via binding of CREB and GATA2 transcription factors. In conclusion, we demonstrate expression of the hOX2R is regulated by a complexity involving a proximal PKA/PKC regulated promoter and a distal promoter regulating tissue specific expression of alternate transcripts which in turn post transcriptionally regulate receptor levels.
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